FI83432C - PROCEDURE FOR DETECTION OF BIOLOGICAL ACTIVITIES. - Google Patents

Description

i 83432

The present invention relates generally to a method and apparatus for detecting biological activity. More specifically, this invention relates to a method for rapidly analyzing materials suspected of containing microorganisms or the like by performing an IR analysis on a sample taken from the gas space of a container in a sample chamber located outside the container 10.

For example, when bacteria are grown in a suitable medium containing a carbon source such as glucose, the carbon source is cleaved to form CO2 during bacterial growth and metabolism. It would be desirable to provide a rapid, sensitive method for analyzing the gas atmosphere evolving above the medium to determine the presence or absence of biological activity.

In many fields, it is important to be able to determine whether or not substances are contaminated with biologically active substances, such as bacteria and the like. Examples of such fields are medicine, food industry, pharmaceutical industry, cosmetics industry and public health work.

It has long been standard practice to place - 25 samples of material from which the presence of biologically active substances is to be examined on a semi-solid medium in a petri dish and to make visual observations of any microbial growth that may result. In another similar procedure, a ste-30 bottle with a liquid medium is inoculated with the material to be examined and again observations of growth are made visually. Such methods are not only slow and laborious, but because they depend on the subjective 35 judgment of individual observers, the result obtained is not uniformly reliable.

2,83432

Methods for detecting bacteria have also been developed by incubating a test material sample in a sealed container with a radiolabeled medium and then examining the atmosphere above the medium in the container to determine whether or not radioactive gases are formed. This type of system is described in U.S. Patent Nos. 3,676,679 and 3,935,073. Such systems are fast and reliable, but suffer from a number of disadvantages, mainly due to the use of radioactive materials. Radioisotope-labeled materials are expensive and require special handling during storage, use, and disposal. In addition, although the amounts of radioactivity associated with the use of such systems are very small, personal fear of radioactivity may deter potential users.

Systems have been described that do not in any way require the use of radioactivity. U.S. Patent No. 4,182,656 describes a method for detecting biologically active materials based on the use of stable carbon-13 enriched media. Although this method eliminates the potential need to use radioisotopes in a detection system, nutrients enriched in carbon-13 are less available and are significantly more expensive than the corresponding radionuclide-labeled compounds. As the molecular properties of 13CO2 are very similar to those of 12CO2, which is the predominant isotopic form of carbon dioxide, and as 13C accounts for more than 1% of the total carbon in the environment, special care should be taken to ensure that 13CO2 is detected primarily at the same time. ignored. The mass spectrometer is commonly used to make observations of changes in the incidence ratios of stable isotopes, and was also used in the development of the above-mentioned patent. Mass spectrometry requires relatively high complexity vacuum equipment and is therefore not a suitable tool for use in a typical microbiological laboratory.

U.S. Patent 4,073,691 describes a non-radio-5 metric means for detecting biologically active substances by making observations of a possible change in the nature of the gas above the liquid medium in a closed bottle system. Changes in the nature of the gas are determined by measuring the ratio between the selected product gas and the inert reference gas, which is also present in the measurements, before and after the bottle is exposed to the conditions that cause the bacteria to grow. The use of an inert reference gas for ratio measurement purposes requires that the detection system react with both the CO 2 released as a result of the metabolism and the inert reference gas, which makes the equipment required for such measurement complex. The concentration of inert gas in the culture gas used in such a system must be known and reproducible from one batch of culture gas to another, which further complicates the detection system.

The use of radioisotopes or stable isotopes or the use of inert reference substances has generally been considered necessary to achieve the detection of small amounts of gas produced by metabolism. Of the various gases produced by bacterial metabolism, carbon dioxide is the gas most commonly produced by various bacterial tribes, yeasts, and other primitive organisms. Thus, there is a need to develop an apparatus system for measuring metabolically generated carbon dioxide for the detection of bacteria and the like that does not require isotope enrichment or labeling of nutrients and is not dependent on the addition of an inert reference gas to the culture flask.

It is therefore an object of the present invention to provide a rapid method for detecting the presence or absence of biologically active substances.

Another object of the invention is to provide a method for detecting the presence or absence of biologically active substances, which method uses relatively inexpensive materials as well as relatively simple equipment.

It is a further object of the invention to provide an instrumental method for detecting the presence or absence of biologically active substances which is not dependent on subjective interpretation.

It is also an object of the invention to provide an apparatus system for detecting the presence or absence of biological activity which avoids the use of isotope-enriched or labeled nutrients or the addition of an inert substance to be used as a reference in the concentration ratio measurement.

It is a further object of the invention to provide an apparatus system which uses IR analysis to detect biologically active substances and which provides the best possible detection sensitivity by matching the carbon dioxide content of the prepared culture vessels with the external culture medium fed to the apparatus for testing, calibration and purification.

These and other objects of the invention are achieved by a method for the detection of biologically active substances, comprising the following steps:

A sealable sterile container, commonly referred to herein as "pul-30 lo", containing a sterile, non-isotopic culture medium is provided. The medium contains a known concentration of dissolved carbon dioxide. The gas above the medium contains an amount of carbon dioxide in equilibrium with the culture medium. The carbon-35 dioxide content in the tank gas space and the carbon dioxide content in the bottled li 5 83432 gas used in the equipment test are approximately equal at a given pH of the medium and at the desired incubation temperature. Bottled gas used for various equipment testing purposes is referred to herein as "culture gas".

A sample of the material whose biological activity is to be examined is placed in a container and the container is closed. The tank is then placed under conditions favorable to normal metabolic processes for a period of time sufficient to effect the formation of gaseous carbon dioxide by the cleavage of various carbon sources in the medium if bacteria are present in the inserted sample.

The gas in the upper space of the flask is then drawn from the flask and circulated to the sample chamber of the IR detection system. through the tank to determine the carbon dioxide content of the gas space. The possible significant increase in the carbon dioxide content of the tank top gas relative to the carbon dioxide content of the bottled culture gas is indicative of biological activity. Without limiting the scope in any way, the method according to the invention is particularly suitable for the detection of medically important bacteria.

If the test sample produces metabolic background activity, such as fresh whole blood metabolic activity, the CO 2 concentration in the culture gas can be adjusted to equal the concentration in the flask gas space achieved at the desired incubation temperature after the addition of the blood sample or other material. The presence of bacteria, charcoal or the like in the sample is then detected as an increase in the CO 2 concentration in the gas space of the flask relative to this normal concentration, which is equal to the CO 2 concentration in the culture gas.

The invention also includes apparatus for carrying out the described method. The equipment includes a tank in which 6,834,32 can be loaded with a sample of material whose biological activity is to be analyzed, and a culture medium containing various carbon sources capable of maintaining normal metabolic processes, one of which results in the production of carbon dioxide. The apparatus also includes means for taking a sample of the gas space of said tank. It also has a device for measuring the carbon dioxide content of a gas by IR analysis. The sampling means and the measuring means are connected to each other by means of a pneumatic system. The equipment has a pump for circulating the headspace gas from the tank through the CO2 detection system back to the tank. The apparatus also includes means for purging the gas treatment system with bottled gas to remove any sample gas debris from the 15 recirculation and metering systems and means for testing the gas handling system to ensure proper operation of the recirculation pump, proper in-line and the appropriate CO2 content of the culture gas.

The device preferably has means for rapidly and sequentially analyzing a plurality of containers to be examined, each containing its own sample of material, the biological activity of which is to be examined. In a preferred type of equipment, the containers are held firmly in place in a rectangular shape by means of a plate designed for this purpose. The apparatus includes means for moving the gas sampling means along the tank row 30 in one direction. In addition, the apparatus includes means for moving the dish in a direction perpendicular to the first said transfer direction so that each vertical row of the dish is reached by the gas sampling means. Gas analysis is performed sequentially on all bottles in a given vertical row 35, after which li 7 83432 is transferred from the plate so that the next vertical row is to be analyzed.

Device control and processing of the results is performed by a microprocessor-based system in which the program is stored in read-only memory (ROM) and the results are stored in read-write memory (RAM). The operator interface is provided by a conventional computer terminal connected to the system using the RS-232 serial communication protocol. A similar RS-232C port is used to communicate with an external computing system.

10 The hardware software allows the user to set device operating parameters, set detection criteria on which positive results are based, register sample containers in the system, obtain test history results, obtain a list of new positive results, test sample containers manually, and perform an automatic program. the use of the service.

The method, apparatus and culture medium according to the invention are characterized by what is stated in the claims.

Figure 1 shows the apparatus used to practice the present invention.

Figure 2 is a schematic diagram of the main components of the apparatus shown.

. Figure 3 is a schematic diagram of a pneumatic system used in the apparatus shown.

Fig. 4 is a timing diagram illustrating a pre-flush cycle of a pneumatic system.

Fig. 5 is a timing diagram illustrating a test sub-cycle of a reservoir of a pneumatic system 30.

Figure 6 is a simplified flow diagram of the electrical system of the apparatus.

In Figures 7-12, the solid line curve represents the average of 9 or 10 inoculated samples and the dashed-35 curve 40 averages the uninoculated reference sample.

Figure 7 is a graph depicting the results of an IR detection experiment performed on the demanding microorganism Neisseria meningitidis aerobically.

Figure 8 is a graph illustrating the results of an IR detection experiment performed on the demanding microorganism Streptococcus pneumoniae aerobically.

Figure 9 is a graph depicting the results of an IR detection experiment performed on the demanding microorganism 10 Haemophilus influenzae aerobically.

Figure 10 is a graph depicting the results of an IR detection experiment performed on a demanding microorganism Streptococcus pneumonia anaerobically.

Figure 11 is a graph depicting the results of an IR-detek-15 test performed on a demanding microorganism

Bacteroides fragilis anaerobically.

Figure 12 is a graph depicting the results of an IR detection experiment performed on the demanding microorganism Bacteroides vulgatus.

The curve drawn through closed circles in Figures 13-26 shows the average of 8 inoculated samples detected by the IR method according to the invention; a curve drawn through closed triangles of the average of 8 transfected samples detected by commercial radiometric detection methods; through open circles and open triangles The plotted curves represent the average of 8 uninoculated reference blood samples detected by IR and radiometric methods, respectively, the limit of detection is indicated by an arrow (- *).

Fig. 13 is a graph showing the results of a kinetic detection experiment comparing the response of an IR detection system to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Escherichia coli.

Fig. 14 is a graph depicting the results of the kinetic detector 9 83432 test comparing the response of the IR detection system to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Klebsiella pneumoniae.

Figure 15 is a graph illustrating the results of a kinetic detection experiment comparing the response of an IR detection system to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Pseudomonas aeruginosa.

Figure 16 is a graph showing the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Staphylococcus aureus.

Figure 17 is a graph showing the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Staphylococcus epidermidis.

Fig. 18 is a graph showing the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Streptococcus faecalis.

Figure 19 is a graph illustrating the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Streptococcus pneumonia.

Figure 20 is a graph illustrating the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Haemophilus influezae.

Fig. 21 is a graph illustrating the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Candida albicans.

Figure 22 is a graph depicting the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the aerobically tested microorganism Neisseria meningitidis.

Figure 23 is a graph depicting the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the anaerobically tested microorganism Clostridium novyii.

Figure 24 is a graph depicting the results of a kinetic detection experiment comparing the response of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the anaerobically tested microorganism Clostridium perfringens.

Figure 25 is a graph illustrating the results of a kinetic detection experiment comparing the results of IR detection to the response of a conventional radiometric detection system as a function of the incubation time of the anaerobically tested microorganism Bacteroides fragilis.

Figure 26 is a graph depicting the results of a kinetic detection experiment comparing the response of IR detection to that of a conventional radiometric detection system as a function of the incubation time of the anaerobically tested microorganism Bacteroides vulgatus.

Fig. 27 is a bar graph illustrating the difference in detection time between two similarly adjusted reservoirs that were kinetically examined to compare IR detection to conventional radiometric detection. The results are given as the time differences between the two systems in the experiment.

. Fig. 28 is a graph illustrating the IR response of the growth of the anaerobic microorganism Bacteroides fragilis compared to the response of a sterile reference sample when the equipment culture gas used does not contain carbon dioxide.

Fig. 29 is a graph illustrating the IR response of the growth of the anaerobic microorganism Bacteroides fragilis compared to the response of a sterile control sample when the equipment culture gas used contains 2% carbon dioxide.

Figure 30 is a graph depicting the IR response of the growth of the anaerobic microorganism Bacteroides fragilis compared to the response of a sterile control when the culture gas used in the apparatus contains 5% carbon dioxide.

Figure 31 is a graph depicting the IR response of the growth of the anaerobic microorganism Bacteroides fragilis compared to the response of a sterile control when the culture gas used in the apparatus contains 10% carbon dioxide.

Fig. 32 is a graph showing the release of carbon dioxide from an anaerobic medium supplemented with bicarbonate as a function of hydrochloric acid addition.

Figure 33 is a graph depicting the response of IR detection to Bacteroides fragilis as a function of incubation time using bicarbonate-supplemented medium and non-supplemented medium.

Figure 1 generally shows a detection apparatus that implements the principles and guidelines of the invention. The containers 1 from which the biological activity is to be determined are arranged on rectangular boards 2 30 which are placed in place on the examination table. The transparent hinged dust cover 3 protects the container from external contamination during the examination and acts as a working surface for the user. The analysis or testing head 4, located behind the user-openable lid 5 35, moves in the y-axis direction of the plate and each measures the carbon dioxide content of the gas space of the successive tank. Once each vertical row of containers has been measured, the plate moves under the test head so that the next vertical row of containers becomes examined. The hardware is microprocessor-based, and all operation control and connections to the 5 users take place via a CRT terminal. Test results and other information of interest to the user are also available on a separate printer (not shown).

The apparatus is particularly useful for early detection of the general presence of most medically significant bacteria in materials such as blood, urine, spinal fluid, synovial fluid, water samples, and the like. The presence of such bacteria is easily detected by measuring the amount of CO 2 released when the analyte is placed in a medium containing one or more carbon sources that are cleaved to form CO 2, and the medium and the sample therein are then incubated. The CO2 content of the gas sample taken from the substrate, which is significantly higher than the CO2 content of the selected culture gas, is an indication of the presence of microorganisms in the original material sample.

Figure 2 schematically shows the main parts of the apparatus. A sample of material to be analyzed, such as blood, urine, or the like, is placed in a sterile culture container 1 equipped with a self-closing rubber septum and an aluminum capsule. The tank has a suitable medium 2, which is buffered so that it remains at the desired pH value and the desired CO 2 concentration is obtained in the gas space 3 above the medium. The container is then incubated under conditions favorable to biological activity. At suitable intervals, the two hollow, pointed steel needles 5 and 6 of the needle device 4 are pushed through the septum of the corresponding bottle. The pneumatic system 10 with the pump 11 circulates the upper space gas of the container 35 through the needle 5, the submicron filter 7, the IR-CO 2

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i3 83432 through the measuring chamber 12, submicron filter 8 and needle 6 of the analyzer 13 back to the flask. The resulting CO 2 reading is digitized and registered in the data processing / control system 14. The results are displayed by means of 5 CRT terminals 15 or printers 16 connected to the data processing / control system.

After each measurement, the needle device is withdrawn from the reservoir, and aerobic or anaerobic culture gas from an external source 17 is used to remove the upper space gas remaining from the needle device, pneumatic system, and IR sample chamber 12 into the measurement system. The needle heater 9 is then moved around the needles 5 and 6 and the needles are heated to a temperature that greatly reduces the possibility of any living organisms remaining on the needles or needles entering the subsequent containers and causing cross-contamination. Testing of a plurality of containers is automatically accomplished by using a plate 18 made so that a plurality of containers 20 can be placed in a rectangular shape. The apparatus has actuating and sensing means which move the apparatus from the plate in the y-axis direction of the examination table. Parts 4-9 form a test head assembly 19 which includes sensing actuating members that move the assembly 25 as a whole in the x-axis direction of the examination table.

Since the CO 2 concentration in the gas space of the sterile bottle at the incubation temperature is determined by the chemical equilibrium during the formulation and filling of the container during preparation, and because the CO 2 content of the external culture gas is chosen to be approximately equal to the CO 2 in the sterile container n equilibrium concentration, a correction for the CO2 content of the gas space can be made by subtracting the value measured for the culture gas 35. The corrected CO2 readings of incubated and measured containers containing sterile material are thus very close to zero, while for containers with biological activity, corrected readings are obtained that are statistically significant higher than the corrected readings of nol-5 la, which are calibrated. so that they resemble readings obtained with a BACTEC radiometric instrument sold by the Johnston Laboratories Division of Becton Dickinson and Company, Coc-keysville, Maryland, designated "growth value unit-10 kö", abbreviated as "GV".

Details of the pneumatic system of the apparatus are shown schematically in Figure 3. Culture gas from an external pressure controlled source used to measure tanks containing aerobic substrate is filtered by a particulate filter F3 and then fed to other parts of the system via an electrically operated solenoid valve V3. The anaerobic culture gas used to examine the tanks containing anaerobic cultures is fed in the same way and filtered as filter F4 20 and its flow is controlled by solenoid valve V4.

The pneumatic resistors R1 and R2 cause flow-dependent pressure drops in both branches of the pneumatic circuit. Pressure sensors PT1 and PT2 are used to measure the operating pressures in each branch of the pneumatic circuit. The readings obtained at the various stages of the operating cycle are used to ensure the proper functioning of the equipment and to provide adequate fault detection in the event of leaks, blockages or pump breakages. Solenoid valves VI and V2 isolate 30 gas space sampling circuits from other parts of the system. The diaphragm pump P circulates the headspace gas in the sampling circuit during the measurement and causes the pressure differences required for flushing and functional testing of the system.

When measuring the CO 2 content of the upper space gas from the tank, the needles N1 and N2 pierce the elastomeric plug 83432 of the culture tank CC. The headspace gas is drawn through the needle N1 through the sterilizing filter / f1 filter and then through the measuring cell SC of the non-dispersive IR analyzer by means of the pump P. The supernatant gas is returned to the NH tank and is intended to help prevent contamination of subsequent culture tanks by non-sterile foreign matter accumulating on gas samples from positive cultures in the sampling needles or needles during inoperative testing of the measurement system or exposure of the needle system to ambient air. The pneumatic system together with the non-dispersive IR carbon dioxide analyzer forms the measuring system of the equipment. A suitable non-dispersive IR analyzer is the Series V CO2 IR Analyzer 15 (Sensors, Inc., Saline M148176). Suitable solenoid valves were supplied by ITT General Controls (Glendale, CA 91201). Pressure transducers suitable for said pressure measurements are available from the MicroSwitch Division of the Honeywell Corporation (Freeport, IL 61032). A diaphragm pump suitable for a pneumatic 20 system consists of a Model N05 pump head (KNF Neuberger, Inc., Princeton, NJ 08540) connected to a P / N DB31D-12 engine (Eastern Air Devices, Dover, NH 03820). Although the selection and installation of the components of the measurement system described herein is suitable for carrying out the present invention, other components and installations may be used, as will be apparent to those skilled in the art.

The examination of the biological activity of the array of sample containers, including functional testing and needle heating, performed by the method described herein constitutes a single test cycle of the apparatus performed by a measurement system and controlled by a data processing system. The entire test cycle consists of a pre-flush sub-cycle for each tank group under test and a tank test sub-cycle performed separately for each tank in group 35. Since the details of the hardware test cycle is 83432 depend greatly on the choice of measurement system components and the structure of the hardware control software, the following description of the test cycle is an example of the various hardware functions required to implement the method using a prototype version of the invention.

Various functional tests of the pneumatic system and the IR analyzer are performed during the test cycle in the data processing system / control system control area. The desire to be able to test all important components of the measurement system is largely the reason for using a relatively complex pneumatic system in addition to the components of the gas sampling circuit (N1, F1, SC, F2, N2 in Figure 3).

The pre-flush sub-cycle is most easily understood by the timing diagram shown in Figure 4 and the diagram of the pneumatic system of Figure 3. At the beginning of the test cycle, the pre-flush sub-cycle begins with the test head and associated needle device raised, thus exposing the ambient room air. All solenoid valves are closed. It is assumed that aerobic tanks are to be studied, so the culture gas is fed through V3 if necessary. Pump P is started briefly so that the sample chamber SC of the IR analyzer is filled with room air flowing through needles N1 and N2. The needle heater NH is also turned on at this stage to preheat the heater for the tests below and to prevent biological contamination of the system by materials accumulated on the exposed needles. The analyzer is allowed to stabilize for a short time; the needle heater is kept around the needle device during this time.

At the end of the stabilization period, the analyzer reading shall be taken and compared to the upper and lower limits of the room CO2 concentration stored in the data processing system 35. A normal reading allows testing to continue; an abnormal value interrupts testing and prompts the user to check the IR analyzer zero setting. At this stage, the valve V3 is opened for a short time, the readings of the pressure sensors PT1 and PT2 are taken and compared with the stored parameters to ensure that the culture gas comes at the correct supply pressure, as well as to check the proper operation of the pressure sensors. The difference between the readings given by the pressure sensors is compared to the stored constant, and the device prompts to service the pressure sensors if the value in the memory is exceeded. The PTlz reading is then compared to another constant pair, which are the upper and lower limits of the reading, and the device warns of too high or low culture gas pressure unless the 15 reading falls within the limits. Testing is interrupted in both cases.

The valve V3 is then closed to save the culture gas, while the heating of the needles continues until the internal temperature of the needles reaches the desired value. The heating of the needles is then stopped, the removal of the heating device is started, the pump P is started and the valves V1, V2 and V3 are opened, allowing the culture gas to flow through the pneumatic system and the IR sample chamber SC and pump P1 to be filled with culture gas. The gas is allowed to circulate for several seconds to ensure that the system is flushed and filled with culture gas. The readings of the pressure sensors PT1 and PT2 are then taken and their difference is compared to a previously stored limit to ensure proper operation of the pump. A difference of 30 within the normal range allows testing to continue; an abnormal separation interrupts testing and causes a prompt to check the pump and connecting piping. The pump P is then stopped, after which the valves V1, V2 and V3 are closed.

The IR analyzer is allowed to stabilize for some ai-35 kaa. The IR analyzer reading is then read, subtracted from the room air reading in the 8 3 432 memory, and the result compared to the normal limits given for the selected culture gas. A normal result within the limits allows testing to continue; an abnormal result interrupts the test-5 test and prompts you to check whether the C02 concentration in the culture gas is either too high or too low and whether the measurement system is working. The CO 2 content of the culture gas is also stored in the memory of the data processing system and is used to correct the measured headspace gas concentration readings of the cylinders according to this method.

The pump P is then started and the valves V1 and V3 are opened with V2 closed, whereby the culture gas flows out through the needles N1 and N2. The reading of the sensor PT2 is then taken and compared with the upper and lower 15s in the memory to ensure that the culture gas pressure is neither too high nor too low. Abnormal readings interrupt the test and instruct to check the pressure of the external gas source, whether it is too high or low. The pump is then stopped, which breaks the connection between the two branches of the system, and valve V2 is opened. With valves VI, V2 and V3 now open, the culture gas flows through each branch of the pneumatic system and out of the needles N1 and N2. The readings of the pressure sensors PT1 and PT2 are again taken and compared separately with the values in the memory to ensure that the pressure drop caused by each needle and filter is within acceptable limits. An incorrect result will interrupt the test and prompt you to check the needles for clogged filters (high pressure) or to check the hose connections (low pressure). Then valves VI, V2 and V3 are closed and the pre-flush sub-cycle ends.

The tank test sub-cycle follows the pre-flush cycle and is repeated for each tank under test. The timing of the apparatus during the test sub-cycle is shown in Figure 5, which refers to the pneumatic system shown in Figure 3. At the beginning of this sub-cycle, all valves are closed and the needle heater is off. The measuring system, which includes needles N1 and N2, the sample chamber SC and the tubes connecting them, contains only 5 culture gases as a result of the pre-flush. The plate and test head are moved so that the first container to be examined comes under the test head, as previously shown in Figure 2. The correct position of the bottle is determined by various optical sensors (not shown) under the control of the data processing system. The existence of an appropriate position in the x- and y-axis directions is the zero moment of the test cycle. The preselected culture gas source is used for either aerobic or anaerobic testing and is compared to a container plate 15 with a code number to ensure that the correct culture gas has been selected. Compatibility leads to continued testing, while incompatibility interrupts the experiment and results in a warning of incorrect culture gas selection.

Valve V3 is opened, allowing the culture gas to enter the system, and sensors PT1 and PT2 are read and their difference is calculated. A difference that exceeds the limit in memory results in a prompt to service the pressure sensors, while a reading of PT1 outside the given upper and lower limits results in a prompt to check whether the pressure of the culture gas 25 is too high or too low. Then valve V3 is closed. The test head is then depressed, allowing the needles N1 and N2 to penetrate the elastomeric plug of the container and open a path to the gas space of the container. The pump P is then started, whereby the headspace gas is transferred to the sampling branch of the pneumatic system.

The closed nature of the headspace gas circuit causes the headspace gas to be diluted with the culture gas in a ratio dependent on the volume ratio of the tank gas space to the sampling branch, which in turn causes the measured CO2 concentration to be different from the gas space 2o 83432 CO2 before sampling. This unavoidable dilution should be minimized to maintain the effectiveness of this test method. Ideally, the volume of the sampling branch is very small compared to the gas volume of the tank, resulting in an insignificant dilution of the upper gas as it circulates through the sampling branch. For practical reasons, the minimum dilution that can be achieved is about 50%, a value that is only achieved by carefully minimizing the lengths of the connecting pipes in a pneumatic system, by selecting solenoid valves with the smallest possible dead volume, and the like. In order to minimize the correction required by the measurement and to maximize the sensitivity of the test with the increase in CO2 activity of the 15 gas spaces caused by biological activity, it is an important aspect of this invention that the CO 2 content of the external culture gas be adjustable to approximately CO2 concentration at incubation temperature before addition of test sample. This is important for the practical implementation of the method presented here.

The carbon dioxide content of the selected culture gas is adjusted to approximately equal to the prevailing carbon dioxide concentration in the gas space of the closed bottle prior to sample addition and incubation. Generally, the anaerobic culture gas contains about 1 to 10% carbon dioxide and about 0 to 10% hydrogen with the remainder being nitrogen. The aerobic culture earth generally contains about 1-10% carbon dioxide with the remainder being air. The concentration of CO 2 in the gas space of the sterile bottle depends on the conditions under which the medium is added and the composition and amount of medium. In a preferred embodiment of the invention, the CO2 content of the headspace gas in a 60 ml flask containing 30 ml of anaerobic medium is about 3-5%. All percentages reported herein are 35 percent by volume, unless specifically stated otherwise 2i 83432. A similar bottle with 30 ml of aerobic medium has a CO 2 content of about 2-3% in the gas space.

The needle heater is switched on and the pump is operated until the complete mixing of the culture gas and the headspace gas in the sampling branch and the culture flask is ensured. The pump is then closed and the IR analyzer is allowed to stabilize while the test head is raised, leaving the needles out of the culture tank. 10 The result from the IR analyzer is then read, processed by a data processing system and stored in memory. Valves VI, V2 and V3 are then closed to flush the upper space sample out of the sample branch. Valves VI, V2 and V3 are closed while the pre-switched needle heating device is placed around the needles and the pump is started to circulate room air through the sample branch. The pump is then turned off for a short time and the needles are allowed to continue heating to promote the removal of any biologically active substance 20 in the needles or needles.

Shortly before the need to stop heating the needles, the pump is started again, after which VI and V2 are opened. The pressure sensor PT2 is then read and the value is compared to the stored limits to ensure the appropriate culture gas pressure. The measurement continues when the reading is normal; an abnormal value above or below the set limits will cause the measurement to be interrupted and prompt to check whether the culture gas pressure is too high or low. Valve V2 is then opened with VI and V3 still open and the pump continuing to run. The culture gas is allowed to flow out through needles N1 and N2. Sensors PT1 and PT2 are read again and the pressure difference is compared with the stored value. A result lower than the stored standard value will cause the measurement to be interrupted and prompt to check the 35 pump and the connected pipes. The pressure readings for PT1 and PT2 are checked and the values are compared to pre-stored readings to ensure that the pipe connections are tight and that there are no blockages in the needles and filters. An abnormal value will cause the measurement to interrupt and prompt you to check the pipe connections at low pressure or if there are blockages when either sensor registers too high a pressure.

V2 is then opened and the pump closed, allowing the flue gas to flow to each branch of the pneumatic system. PT1 and PT2 are read again and their readings are compared to the stored values to ensure that the pressure drop across each needle and associated filter is within acceptable limits. The results of the non-standard 15 paints result in an interruption of the examination of the tanks and a prompt to check all pipe connections at low pressure or to check the needles and filters for blockages at high pressure. All valves are then closed and the sampler is started to move the test head and / or plate to a suitable location to examine the next tank in sequence, after which the tank test sub-cycle begins again.

The control of the measuring system, the movement of the plate and the movement and operation of the test head takes place by means of a data processing / control system together with the necessary electronics, as shown schematically in Fig. 6. The main current is standardized as a filter 1 in accordance with domestic and foreign radio frequency difference guidelines. The filtered 120 V AC is supplied to the main power supply 2, 30 to the AC control circuit 3 and the needle heater power source 4. The filtered 120 V AC is also used as a power source for the CRT terminal and printer. The logic level signals from the data processing / control system 5 control the 35 AC motors, solenoid valves connected to the measuring system 6.

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23 83432 and the like, the plate moving device 7 and the test head 2 via the semiconductor switches in the AC control circuit.

The power source of the needle heater provides direct current to prevent a delay in the heating power of the needles as the resistance of the heating parts increases as they age. The heater on and off commands are given by a control system that also monitors the fault signal from the heater power supply to ensure proper operation of the heater. The control system also controls the solenoid valves and the operation of the sampling pump and accepts analog readings from the pressure sensors and the IR analyzer as described above. All analog signals are processed by a multi-channel analog-to-digital converter before the signals are processed. The movements of the dish are controlled by optical sensors that determine the position of the dish and verify the dish code to ensure that the selected aerobic or anaerobic gas is correct for the dish being examined. The UV lamp used to irradiate the bottle stopper 20 is also computer controlled and is on only during testing of the bottles with the appropriate protective cover closed to protect the user from unnecessary UV radiation.

The raw results from the IR analyzer, which are proportional to the IR transmittance in the CO 2 absorbance range, are fed to the data processing system / controller in analog form, digitized, and then linearized to represent the carbon dioxide concentration using Table 30 in the system software. The linearized results are then scaled to provide the user with a reasonable numerical value range ranging from 0 to 200 to 300 arbitrary units, designated the Growth Value Unit (GV). The maximum value of the scaled results depends on whether the aerobic or anaerobic culture gas is used and what proportion of the dynamic range of the IR analyzer is used by the CO2 in the culture gas. Thus, the highest possible growth difference is smaller when studying anaerobic cultures because the CO2 concentration is higher in the 5 anaerobic culture gas.

The entire operating system software is stored in read-only memory (ROM), while the system's fast working memory and storage of results are in random access memory (RAM). The system operates under periodic control with a real-time clock to monitor the timing of sampling and measurement. Critical data and the contents of the system memory are stored and the operation of the real-time clock is maintained in the event of a power failure for at least one week by means of a nickel-cadmium backup battery. The equipment has a power-15 open / reconnect circuit to ensure proper shutdown of the equipment in the event of a power outage. Crucial System and Computer Standards are retained after a power failure is detected and are used to restart the system when the main power is restored. 20 All user communication with the hardware is via an external CRT terminal via menus (menu driven screen presentations). The apparatus has separate menus for display selection, setting of operating parameters, selection of positive result detection criteria, storage of sample containers in the system, obtaining the result history of a pair of aerobic and anaerobic flasks expressed by previously measured growth values. to perform daily hardware maintenance.

To ensure that the hardware meets the performance requirements of clinical use, the operating software also includes self-testing capabilities, including automatic 35 self-testing routines for ROM and RAM,

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25 8 3 432 To check the status of the computer (CPU), supply voltages and backup battery. These routines are implemented with the equipment free between tank tests. Functional checks are also performed on the optical sensors, motors, the needle heater power supply / needle heater and the measurement system. As part of the user’s daily inspection program, detailed functional checks are also performed on the controller, measurement system, power supplies, optical sensors, and various motors. The above-mentioned user inspection procedures provide for the isolation of faults at the sub-level of the equipment.

The media used in connection with the apparatus described above for carrying out the method according to the invention are formulated for the growth and detection of aerobic, facultative and anaerobic microorganisms. It is clear that oxygen is absolutely necessary for the growth of aerobic microorganisms and that the amount of oxygen must be kept to a minimum in order to grow absolute anaerobes. If light-requiring or intolerant microorganisms are of interest, light should be provided or eliminated.

Typical media usually contain water, a carbon source, and a nitrogen source. The carbon source may be a carbonate hydrate, amino acid, mono- or dicarboxylic acid or a salt thereof, polyhydroxy alcohol, hydroxy acid, carbon dioxide / bicarbonate, or other metabolizable carbon or carbon dioxide compound. Some microorganisms assimilate carbon dioxide during growth. Some of these 30 microorganisms require relatively high concentrations of carbon dioxide in the medium because of the low affinity of these microorganisms for carbon dioxide. The carbon source usually contains at least one sugar such as glucose, sucrose, fructose, xylose, maltose, lactose, etc. Also amino acids such as lysine, glycine, alanine, tyrosine, threonine, histidine, leucine, etc. form part of the carbon source of the growing medium. The nitrogen source may be nitrate, nitrite, ammonia, urea or any other assimilable organic or inorganic nitrogen source. The amino acid or a mixture thereof can serve as both a carbon and a nitrogen source. Nitrogen should be present sufficiently to ensure cell growth and replication.

Various calcium, potassium and magnesium salts, including chlorides, sulfates, phosphates, and the like, can be added to the medium. Because such materials are common in microbiological media, the choice of materials and their interrelationships are well known in the art.

The so-called trace elements, which are present in very small amounts, usually include manganese, iron, zinc, cobalt and possibly other elements.

Examples of known growth media that can be used in the context of this invention include peptone broth, trypsin-cleaved soybean broth, nutrient broth, thioglycollate broth, and brain-heart infusion broth. Trypsin-cracked soybean broth-based media (media 6B and 7C for aerobic and anaerobic culture, respectively, sold by Johnston Laboratories, BB1 Microbiology Systems Division 25 of BD, Towson, MD 21204) have been found to work well.

It is well known that most biologically active species cannot function in strongly acidic or basic environments. Similarly, in order for the method and apparatus of the invention to effectively detect carbon dioxide resulting from the metabolism of microorganisms in the form of an increase in CO 2 in the culture vessel gas, it is preferred that the medium be buffered to a certain pH and carefully monitored to ensure proper the carbon dioxide-.35 sidi-bicarbonate equilibrium is formed in the medium and 27 8 3 432 is maintained in it before the inoculation of the medium with the test samples. Suboptimal pH values result in poor CO2 release from the liquid medium, while suboptimal substrate pH values result in too high a CO 2 concentration in the upper space gas to mask the metabolically produced carbon dioxide. Suitable buffers, such as potassium or ammonium phosphates, sodium citrate or the like, can be used to adjust the pH, while various carbonate and bicarbonate salts, as well as gaseous carbon dioxide added to the gas space during the manufacture of the filled culture flask, are most preferably used to detect chemical equilibrium. Since many bacterial species, especially those belonging to anaerobes, require significant concentrations of dissolved carbon dioxide in the medium to grow optimally, the above-mentioned attention to both pH adjustment and carbon balance synergistically promotes both growth and detection.

It should be noted that not all microorganisms 20 metabolize carbohydrate and / or amino acid substrates in the medium to produce sufficient amounts of carbon dioxide to be detected by this method. This is particularly the case for certain anaerobic microorganisms, especially those belonging to the genus Bacteroides, such as B. fragilis. However, the majority of such microorganisms produce significant amounts of various volatile and non-volatile organic acids as substrate cleavage products.

This method provides the detection of these microorganisms by detecting a decrease in the pH of the medium as a result of acid production, as measured indirectly by an increase in the CO 2 concentration in the tank gas space as the carbonic acid-bicarbonate equilibrium changes, which is inevitably related to pH 35. the change. Such indirect metabolism detection 28 83432 makes it possible to detect the growth of a wide variety of microorganisms and metabolism patterns in the gas space through metabolically formed carbon dioxide. The basic importance of the medium for the method and equipment is thus based on a carefully selected bicarbonate content, which both promotes the growth of organisms that require bicarbonate for food and allows the detection of acid-producing organisms that produce little or no metabolic carbon dioxide. Together with any 10 suitable buffer systems, this added bicarbonate increases the carbon dioxide content in the gas space and requires a corresponding increase in the carbon dioxide content of the culture gas. As these concentrations increase, the detection sensitivity of metabolically produced carbon dioxide decreases.

There are two reasons for the decrease in sensitivity. When the CO2 / bicarbonate content of the medium is relatively high, the difference between the carbon dioxide content of the culture gas and the initial carbon dioxide content in the gas space tends to obtain a relatively high absolute value. The difference between crop gas and headspace gas can be at most 20% carbon dioxide at most. When a carbon dioxide analyzer is used at these higher concentrations, it must have a larger measuring range and there is a tendency for errors to occur due to a larger absolute error in the carbon dioxide concentration. The optimum bicarbonate content of the medium for the desired group of organisms is thus the lowest possible concentration which suitably promotes the growth of the organisms and causes the detection of acid-producing organisms. When these choices are made as described in connection with a preferred embodiment of the present invention, all pathogenic bacteria commonly found in blood cultures can be detected rapidly, and the time required for detection does not increase significantly with bacteria that could be detected with much less or no bicarbonate.

li 29 83 432

Generally speaking, a medium suitable for delivering assimilable carbon dioxide as well as sufficient carbon dioxide to detect acid-producing organisms contains an effective amount of carbon dioxide precursors that can form carbon dioxide as a result of the metabolism of the microorganism in the material sample. The precursor is activated as a result of acid formation during the metabolism of acid-producing organisms. The precursor is present in the medium in an amount corresponding to a bicarbonate-10 road equivalent concentration of about 0.5 to 20.0 mmol / l, preferably about 1.0 to 10.0 mmol / l. Suitable precursors include sodium bicarbonate, dissolved carbon dioxide, sodium carbonate and other bicarbonate salts.

At the beginning of the testing process, the medium is inoculated with 15 samples of material to be tested, while maintaining the pH of the medium at a value of about 6.5 to 8.0, preferably about 7.2 to 7.5. The CO 2 concentration in the gas space is about 2.0 to 3.0% in the case of an aerobic medium and about 3.0 to 5.0% in the case of an anaerobic medium and is subject to chemical changes in its equilibrium, volume and temperature, such as described above. The amount of sample used can vary widely, but should preferably be about 1.0 to 20.0% of the volume of medium. After a short delay, any microorganisms present in the medium grow and replicate, followed by a decrease in the rate of reproduction. In addition, the rate of CO2 release will vary depending on factors such as nutrient composition, pH, temperature, inoculum content, and the type of microorganisms present.

For efficient bacterial metabolism, the temperature of the medium containing the sample should be maintained at about 35-37 ° C. For some microorganisms, optimal growth occurs at 20 ° C or cooler, while some may grow best at 45 eC or warmer. In the context of the present invention, any temperature best suited to each situation is 30 ° 83432, provided that attention is paid to the CO 2 concentration in the gas space of the tanks maintained at the desired temperature and the CO 2 content of the culture gas used is adjusted as close as possible. Although satisfactory growth of microorganisms is usually achieved without agitation of the inoculated culture in the tanks, the culture is preferably performed using active shaking, agitation, or the like, which is effective enough to ensure proper release of CO 2 from the medium.

In a preferred embodiment, external mixing is provided by a rotary shaker which forms a vortex on the liquid medium.

To now discuss in more detail the practical aspect of the method carried out by means of the apparatus 15 shown in Fig. 1, the total volume of the culture tanks 1 is preferably 30 to 150 ml, of which the proportion of medium and test sample is 2 to 100 ml. The volume of blood, urine or other sample may be, for example, 0.1 to 10.0 ml. In a preferred embodiment, the culture vessels have an overflow volume of about 60 ml and are loaded with 30 ml of medium and optionally 3-5 ml of sample. The gas space thus accounts for about 50% of the total volume of the tank. It is preferred that the gas space in the tank be about 30-60% of the total female volume of the tank. It is somewhat more important that the ratio between the gas volume of the tank and the volume of the measuring system is kept as high as possible in the light of the above considerations in order to maintain the detection sensitivity of the system.

In order to determine the applicability of the apparatus and method disclosed herein for detecting the presence of biological activity in simulated blood cultures by analyzing the culture vessel headspace gas, a prototype apparatus having the essential features of the apparatus shown in Figure 1 was fabricated for research purposes. The pneumatic system used was approximately as shown in Fig. 12. A commercial IR analyzer (Model AR500R Infrared Gas Analyzer, Anarad, Inc., Santa Barbara, CA 93105) was used to detect CO 2, while the tanks were tested on a modified commercially available BACTEC Model 225 (Johnston Laboratories). The system was controlled by a microcomputer Cromeco System Three (Cromeco, Inc., Mountain View, CA 94040). The operation of the equipment and the test sequence were approximately as described above.

10 In order to simulate the low inoculum concentrations observed in clinical blood cultures and to achieve statistically significant results, however, the inoculations used in the simulated blood culture experiments were prepared so that about 100 colony forming units (CFU) entered the reservoir. The organisms used for the experiments were taken from agar or broth cultures incubated overnight. For standardization, several colonies from agar were suspended in 5.0 ml of thioglycollate broth (anaebrobit) or trypsin-digested soy broth (aerobes) (both supplied by BBL Microbiology Systems, Cockeysville, MD 21030) in a 16 x 125 mm screw cap tube and turbid. to correspond to a transmittance of 57-63% on a Spectronic 88 spectrophotometer (Bausch and Lomb, Rochester, NY 14625) at 600 nm. Alternatively, 0.7 ml of cloudy broth from a BACTEC flask (Johnston Laboratories) was mixed with 5.0 ml of broth and the turbidity was adjusted as above. The standard broth was diluted 1: 100 three times with a similar broth and 0.5 ml of the final dilution (approximately 100 CFU) was added to each flask to be examined in the apparatus. Transfer levels were checked by counting from the dish.

Blood used in kinetic growth experiments was provided by the Community Blood and Plasma Service (Baltimore, MD 21231). This blood, sometimes referred to herein as "storage blood," was taken to order in specially prepared sachets containing 32 8 3 432 sodium polyanethole sulfonate (SPS) as anticoagulant at a final concentration of 0.05%. Kinetic experiments using Neisseria meningitidis were performed with fresh blood because SPS potentially inhibits the growth of N. meningitidis. Growth studies using other demanding microorganisms were similarly performed using fresh whole blood.

All media used in the following examples were provided by Johnston Laboratories. All anaerobic microorganisms were cultured in anaerobic medium 7C 10 BACTEC, which was purged with anaerobic culture gas during the experiment, but was not shaken. This gas contained 2% carbon dioxide and 5% hydrogen with the remainder being nitrogen. Aerobic microorganisms were grown in medium 6BM (medium 6B supplemented with stirring magnets) which was stirred with a magnetic stirrer in a prototype device. The aerobic culture gas contained 2.5% carbon dioxide with the remainder being air. The incubation temperature was adjusted to 35-37 eC in all experiments.

Example 1 Five slow-growing, demanding microorganisms were tested in blood-supplemented medium. Inoculation was performed for each microorganism / broth combination with each vial containing fresh whole blood from a different person. The amount of transfer was 8 to 150 CFU / bottle. Ver-25 storage containers were prepared using fresh whole blood but no microorganism inoculum. The reference tank results were summed to calculate means and standard deviations. The containers were inspected after inoculation and once daily thereafter. The limits of detection were chosen to be 19 GV for aerobic cultures and 34 GV for anaerobic cultures based on experiments to determine the worst possible background values for blood metabolism. The error bars in the following figures correspond to the mean, minus 35 times the standard deviation (SD), for both test and comparison results.

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Figure 7 shows the growth values as a function of the time taken for the experiment for the microorganism Neisseria meningitidis. Detection as a result of CO2 release is observed to occur on day 2 of the experiment. Test and 5 comparison results differ when using +/- 2SD at the end of the experiment as a criterion. Figure 8 shows similar results obtained with Streptococcus pneumoniae. Moderate detection is achieved after 24 hours of incubation. The results obtained with Haemophilus influenzae are shown in Figure 9. Although this microorganism is a weak carbon dioxide producer, detection occurs on the second day of the experiment. The culture results obtained with Streptococcus pneumoniae are shown in Figure 10. A relatively weak, short-term response is observed, but the microorganism is clearly detectable on the second day of the experiment.

Demanding anaerobic microorganisms were studied in the same manner. The results obtained with Bacteroides fragilis are shown in Figure 11. Detection occurs on the second day of the experiment. Detection results for Bacteroides vulgatus show significant dispersal, but the organism is detectable by day 5 in all samples examined, as shown in Figure 12.

Example 2

Kinetic growth studies were performed on a number of clinically significant microorganisms by measuring similar reservoir groups with a prototype IR instrument and an unmodified radiometric instrument BACTEC Model 225. Experimental parameters for kinetic experiments are given in Table 1. Stock blood containing 0.05% SPS was used. for all other microorganisms except Neisseria meningitidis for which freshly drawn blood was used. The points in the following figures are the averages obtained from several experimental measurements as a function of incubation time at 37 ° C. Arrows indicate the limit of detectability-35 in different experiments. The results obtained with uninoculated, blood-supplemented broth are also presented as the averages of several 34,83432 measurements, the measurements were performed at the same time intervals as for micro-organism-inoculated, blood-supplemented media. The containers were measured every two hours for fast-growing microorganisms and every five hours for slow-growing microorganisms.

Table I

Feasibility study of IR detection 10 Experimental parameters of kinetic growth experiments

Parameter Aerobic_Anaerobic_

Culture medium 6BM

(magnetic-

mixture) 7C

Culture gas 2.5% (X 2, final 2% CH 2, 5% H

part air rest N

Test interval fast fast

growing: 2 h growing: 2 H

require: 3 H slow 20 growing: 5 h

Duration of the experiment: fast growing fast: 18-24 h growing: 18 h demanding: 36-45 h slow growing: 70 h 25 Limit of detection, IR 19 34

Limit of detection, BACTEC 20

All exams:

Blood: 5.0 ml stock blood, 0.05% SPS

incubation

temperature: 35 to 37 eC

Transfer 50-100 CFU / flask, standardized spectrophoton: meter; actual numbers determined by 35 counting from the dish

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35 83432

The kinetic results recorded, which compare the detection of the microorganism Escherichia coli by IR detection and radiometric detection, are shown graphically in Figure 13. The detection time 5 is approximately the same for both methods. Figure 14 shows similar results for Klebsiella pneumoniae. Also in this case, the detection times of the IR method and the conventional radiometric method turn out to be approximately the same. For the radiometric 10 BACTEC results, the flat curve at the top represents a reading that meets the full scale of the instrument. Pseudomonas aeruginosa is detected somewhat more slowly by the method of the invention and gives both a stronger and faster response when using the radiometric method 15, as shown in Figure 15. In the case of radiometric results, a flat curve at the top reflects a result that meets the full scale of the device. The responses obtained with Staphylococcus aureus are shown in Figure 16. Detection is approximately simultaneous with both systems, but a stronger response is observed with IR detection. Similar behavior is observed for Staphylococcus epidermis, as shown in Figure 17. In this case, IR detection gives an earlier detection and a more positive response than a radiometric system. The microorganism Streptococcus faecalis was examined in the same manner, and the results shown in Fig. 18 were obtained. IR detection is faster and more potent than radiometric detection for this microorganism. The results obtained with Streptococcus pneumoniea are compared in Figure 19. The advantage of the IR method according to the invention in detection is clearly apparent. Haemophilus influenzae gave the responses shown in Figure 20 when examined. The limit of detection of the microorganism was exceeded by the radiometric method slightly more strongly and faster 35 than by the IR system. Detection of the yeast Candida albicans occurred slightly faster on radiometric examination of 36,83432 and the achieved reading filled the entire scale. Both systems detected this microorganism in approximately the same manner, as shown in Figure 21. Neisseria meningitidis gave the responses shown in Figure 22 in the comparative-5 study. Although both systems detected the microorganism well enough, the BACTEC method provided somewhat earlier and more positive detection.

Anaerobically grown organisms were also studied for detection kinetics. Both systems detected the microorganism Clostridium novyii equally well, as shown in Figure 23. Similar results were obtained for Clostridium perfringens, as shown in Figure 24. The time required for detection is slightly shorter for IR detection. Bacteroides fragilis elicited the response shown in Figure 25. BACTEC detection provides faster detection and a stronger response. The situation is the same for Bacteroides vulgatus, as shown in Figure 26.

The results of the kinetic experiments are shown in Table 2. Based on the kinetic experiments performed on similar plates, IR detection provides a roughly equivalent detection of potentially pathogenic microorganisms selected for study. Further analysis of the kinetic results based on the results of the individual flasks obtained for each microorganism, rather than the average result, showed that the detection times obtained with the IR method of the invention were as good or better than those obtained with the conventional BACTEC radiometric method in 72% of experiments. In approximately 93% of the experiments, detection occurred one test interval later or faster than BACTEC. Test intervals ranged from two hours (fast-growing organisms) to five hours between measurements. '. ’35 tini (slow growing). The kinetic results are reported by the number of test intervals elapsed for detection in Figure 27.

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Table 2 Comparison of IR and BACTEC detectors for different organisms - pairs of bottles made similar 5 5.0 ml stock blood - 0.5% SPS Aerobic organisms -

start with 6BM

Average time to detection (h) _

Organ Experiment Transfer IR Bactec Difference 10 _lkm_ (CFU / flask) _ E.coli 8 (2 h) 62-71 10 10 0 K.pneumoniae 8 62-74 10 10 0 P.aeruginosa 14 53-67 15 14 -1 S .aureus 15 4-169 12 13 +1 15 S.epidermidis 4 1-57 16 18 +2 S.faecalis 8 75-87 12 12 +1 S.pneumoniae 10 (2 h) 18-58 18 20 +2 H. influenzae 10 -250 20 17 -3 C.albicans 11 4-19 31 33 +2. . 20 N. meningitidis 8 82 19 19 0

Aerobic organisms - platform 7C

C.perfringens 9 (2 h) 25 10 12 +2 C.novyii 8 21 10 10 0 25 B.fragilis 9 (5 h) 90 29 25 -4 B.vulgatus 15 (3 h) 100 33 28 -5

Positive value * IR better Negative value = BACTEC better 30 2Determination limits: IR, aerobic - 19 IR, anaerobic * 34 BACTEC = 20 (both) 35 38 83432

Example 3

In order to demonstrate the advantage in detection sensitivity obtained by adjusting the carbon dioxide content of the culture medium of the apparatus to equal to the CO . Control and test tanks containing 5.0 ml of thief-10 companions were prepared. Approximately 100 CFU of test microorganism, prepared, diluted, and standardized by calculation as described in the previous examples, was also added to the test vessels. Culture gases containing 0, 2, 5 and 10% carbon dioxide, 5% hydrogen and the remainder nitrogen were used. Six pairs of flasks, each with a sample and control flask, were tested anaerobically in an IR prototype incubator at 35 ° C for 48 h in each experiment on the culture gas mixture to be tested.

The results obtained for the carbon dioxide-free culture gas are shown in Figure 28. Since the readings are recorded as the difference between the CO2 concentration in the tank headspace gas and the corresponding culture gas concentration, a positive shift is observed for all readings, which inevitably increases the detection limit. When the CO2 concentration of the culture gas is adjusted to equal to the prevailing concentration in the gas space, the bottom values are no longer elevated and the best possible detection is achieved, as shown in Fig. 29. The results obtained with the 5% CO 2 mixture are given in Figure 30. The large negative shift at the beginning could prevent the detection of rapidly growing microorganisms. In addition, incubation for about 15 h and associated repeated testing are required to bring the CO 2 concentration in the gas space into equilibrium with the CO 2-35 concentration in the culture gas. The decrease in the difference between the growth values of the sample and control tanks is clearly noticeable. Figure 31 shows the results obtained with 10% carbon dioxide in the culture gas. The initial negative shift is now much larger and the detection sensitivity is very weak. In addition, incubation for about 36 hours and several repeated measurements are required to bring the CO2 concentration of the headspace gas into equilibrium with the culture gas.

For proper operation of the method disclosed herein, it is preferred that the carbon dioxide content of the culture gas used in the apparatus be adjusted as close as possible to the CO 2 concentration in the gas space of the sterile, non-inoculated containers used in connection with the apparatus. In this optimal situation, the best possible detection is obtained, and the time taken for the upper space gas of the culture tank to equilibrate with the culture gas of the equipment becomes as short as possible. Bottom readings remain stable throughout the incubation period, making it possible to select a limit of detection that is independent of time.

Example 4

It is well known that some clinically significant microorganisms, especially those belonging to anaerobes, produce little carbon dioxide as a result of metabolism. However, the majority of these microorganisms produce significant amounts of volatile and non-volatile organic acids, which are secreted into the medium in an effort to lower its pH. Therefore, experiments were performed to determine whether the chemical balance between carbon dioxide and bicarbonate could be used to detect these microorganisms by measuring the carbon dioxide released from the medium as a result of the change in chemical balance caused by the lower pH of the medium. Indirect CO2 release was first examined in medium 7C supplemented with 16.6 mmol / l sodium bicarbonate. To 0.1 ml HCl containing medium and bicarbonate and to control flasks containing medium alone were added 0.5 M HCl in 0.1 mL portions. 0.1 ml of acid was added to one pair of flasks, 0.2 ml to the next, etc. Similar flask-5 pairs were prepared for pH measurement. CO 2 measurements were performed on the pairs of flasks with equipment, and the released CO 2 was plotted as a function of acid addition in Figure 32. The detection of chemically released carbon dioxide can be clearly demonstrated by comparing the growth value readings of the test and control flasks. The response to a decrease in pH compensates well for the increase in the initial concentration of CO2 in the gas space.

The indirect detection system was then tested with a strain of Bacteroides fragilis that is known not to produce significant amounts of carbon dioxide as a result of metabolism. 16.6 mmol / l bicarbonate was added to medium 7C (Johnston Laboratories). Similar control flasks without bicarbonate were also prepared. To each flask was added 5.0 ml of the stock blood described above; the test flasks were inoculated with about 100 CFU of the microorganism. The results of the test are shown in Figure 33. In the case of a medium supplemented with bicarbonate, a greatly improved detection of the micro-organism is observed. Although blood-induced background readings increase somewhat, the benefit of the availability of additional carbon dioxide is clearly noticeable.

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Claims (19)

A method for detecting the presence of microorganisms in a sample of a material to be analyzed, characterized in that it comprises the steps of: a) introducing the sample to be examined in a sterile vessel containing a a culture medium and a gas space located thereon, the culture medium having a carbon source and the original CO 2 concentration in the gas space present above is known; b) incubating the vessel under conditions favoring the metabolism of the microorganisms; c) introducing at the analysis end a culture gas whose CO2 concentration is substantially the same as the original known CO2 concentration in the gas present above; d) determines the CO 2 concentration of the culture gas at the analysis end, which includes sensing and moving means used in sampling the above-existing gas in the vessel; e) establishes a flow link between the space provided and the analysis end; f) mixing the culture gas at the analysis end with the gas located above; G) measures the CO 2 concentration in the mixture of the culture gas and the gas present above at the analysis end; and h) compares the measured CO 2 concentration of the mixture with the CO 2 concentration of the culture gas determined in step d) to detect the presence of microorganisms. Method according to claim 1, characterized in that the CO 2 concentration level in the culture gas which is mixed with the sample of the gas present above is approx. 1 - 10%. 35 3. A process according to claim 2, characterized in that the CO 2 concentration level in the culture gas 8 is approx. 2 - 5%. 4. A method according to claim 1, characterized in that a measuring cell is provided in the sensing end 5 of the analyzing end, and the step of establishing a flow connecting link allows the measuring cell to measure the CO 2 concentration of the mixture. 5. A method according to claim 4, characterized in that the flow connecting line 10 is rinsed with culture gas before step d). 6. A process according to claim 5, characterized in that the CO 2 concentration level of the culture gas is adjusted to approx. 1 - 10%. 7. A method according to claim 1, characterized in that whole blood is used as analytical sample. Method according to claim 1, characterized in that several analysis vessels are analyzed one after the other. Apparatus for use in the method according to claim 1, for analyzing material vis-a-vis the presence of biological activity by infrared absorption, characterized in that said apparatus comprises: a closed container with a microorganism intended culture medium which includes a carbon source which can produce carbon dioxide by metabolism of a microorganism present in a sample of the material, the closed container above the culture medium having a gas space containing a gas located above, wherein carbon dioxide is present in equilibrium means for introducing a sample to the material in the container, means for exposing the container to the sample and the medium for conditions which favor a normal metabolic process of microorganisms possibly present in the sample to increase the carbon dioxide content of the above gas. a value li 47 83432 above the equilibrium level of the above gas, means for removal a sample of the above gas and for introducing the sample into a sample cell, means for generating an infrared ray bundle, and means of IR detection for detecting the IR absorption of the above gas in the sample cell. 10. Apparatus according to claim 9, characterized in that the sealed container is provided with a permeable lid. 11. Apparatus according to claim 10, characterized in that said means for removing a sample of the above gas and for introducing the sample into a sample cell comprises a first needle in flow connection with an inlet to the sample cell and a second one. flow in connection with an outlet from the sample cell. 12. Apparatus according to claims 10 or 11, characterized in that it includes means for inserting the first and second nails through the permeable lid to form a closed loop for circulating the above-located gas. 13. Apparatus according to claim 12, characterized in that it includes a pumping device for effecting circulation of the above gas. 14. Apparatus according to claim 12, characterized in that it includes means for introducing cultured gas into the loop prior to insertion of the first and second nails into the permeable cap. 15. Apparatus according to claim 14, characterized in that the culture gas has a carbon dioxide content which is substantially equivalent to the equilibrium content of carbon dioxide in the above gas. 16. Microorganism culture medium to be used in the method of claim 1, characterized in that it comprises a bacteriological nutrient and an effective amount of a carbon dioxide precursor from which carbon dioxide can be produced by metabolism of a microorganism present in a material sample and which precursor is activated by the generation of acid during the metabolism of a microorganism which is present in a sample introduced into the culture medium. The culture medium according to claim 16, characterized in that the precursor is present in an amount sufficient to form about 0.5-20 mmol of equivalent bicarbonate per liter of culture medium. The culture medium according to claim 16, characterized in that the precursor is present in an amount sufficient to form about 1 - 10 of equivalent bicarbonate per liter of culture medium. The culture medium according to claim 16, 17 or 18, characterized in that the precursor is sodium bicarbonate, dissolved carbon dioxide, sodium carbonate or some other bicarbonate salt. II